Epitope for switching to TH2 cell and use thereof

ABSTRACT

The present invention relates to a novel epitope to convert T cell to type 2 helper T (TH2) cell. Specifically, the present invention relates to an epitope constituting the 20th to 30th amino acids (SEQ ID No.2) of extracellular domain (ECD) of activation-inducible tumor necrosis factor receptor (AITR), an antibody recognizing the epitope, a polynucleotide encoding the epitope, a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide encoding the epitope or antibody, a transformant introduced with the vector, a composition comprising the antibody for converting T cell to TH2 cell and a method of conversion, a pharmaceutical composition comprising the antibody for preventing or treating autoimmune disease, and a method for treating autoimmune disease using the antibody.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/739,763, filed Jun. 15, 2015, which is a divisional of U.S.patent application Ser. No. 13/861,675, filed Apr. 12, 2013, and whichclaims priority from Korean Application No. 10-2012-0061791 filed onJun. 8, 2012, and Korean Application No. 10-2012-0088647 filed on Aug.13, 2012, all of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “2012994-0028_SL.txt” onSep. 13, 2018). The .txt file was generated on Aug. 6, 2018 and is25,845 bytes in size. The entire contents of the Sequence Listing areherein incorporated by reference.

TECHNICAL FIELD

The present invention relates to a novel epitope to convert T cell totype 2 helper T (T_(H)2) cell. Specifically, the present inventionrelates to an epitope constituting the 20^(th) to 30^(th) amino acids(SEQ ID No.2) of extracellular domain (ECD) of activation-inducibletumor necrosis factor receptor (AITR), an antibody recognizing theepitope, a polynucleotide encoding the epitope, a polynucleotideencoding the antibody, an expression vector comprising thepolynucleotide encoding the epitope or antibody, a transformantintroduced with the vector, a to T_(H)2 cell and a method of conversionfor the same, a pharmaceutical composition comprising the antibody forpreventing or treating autoimmune disease, and a method for treatingautoimmune disease using the antibody.

BACKGROUND ART

Various types of receptors belonging to the tumor necrosis factorreceptor superfamily (TNFR) superfamily are involved in the regulationof cell proliferation, cell differentiation, and cell death. Also, TNFRreceptors play an important role in development of immune response,tumor necrosis, and protection against autoimmune diseases. Examples ofthe TNFR receptors include CD28, 4-1BB, OX40, CD40, or CD27.

Human activation-inducible tumor necrosis factor receptor (AITR) is amember of TNFR superfamily, and is also called glucocorticoid-inducedTNFR-related protein (GITR), tumor necrosis factor receptor superfamilymember 18 (TNFRSF18), or CD357. AITR is a type I transmembrane proteinof TNFR superfamily. Immunoregulatory T cell (T_(reg) cell orCD4⁻CD25^(high) T cell) constitutively expresses AITR at high level, andwhen peripheral blood mononuclear cell (PBMC) is activated, expressionof GITR is rapidly up-regulated. In particular, a signal transductionthrough AITR is known to inhibit a suppressive function of T_(reg)cells, which in turn increase a resistance of CD4⁺ and CD8⁺ T cells tothe suppression by T_(reg) cells, thereby activating the CD4⁺ and CD8⁺ Tcells. Also, a monoclonal antibody (mAb, e.g., DTA-1) that specificallybinds to GITR or a physiological ligand thereof i.e., GITRL is known toenhance antitumor immunity through various in vivo models. On the otherhand, activation of GITR inhibits T_(reg) cell which is capable ofsuppressing the autoreactive T cells, thereby promoting autoimmuneresponse.

Although there has not been much information found about the biologicalfunctions of AITR and AITR ligand (AITRL), it is known that AITR isexpressed at basal level in a resting T cell, but its expression levelrapidly increases upon activation of T cell. Also, it has been reportedthat AITR expression level was increased in herniated disc tissue cellisolated from lumber disc herniation patients as well as in the T_(reg)cells from active systemic lupus erythematosus patients.

AITR binds with TNFR-associated factors (TRAFs) such as TRAF1, TRAF2 andTRAF3. When AITR interacts with its ligand, this recruits TRAF2 andultimately activates NF-KB/NIK (Kwon, B. et al., J Biol Chem 274,6056-6061, 1999). TRAF protein is a type of cytoplasmic adapter protein,and is involved in signal transduction for extracellular proliferation,differentiation, activation and migration. The N-terminal sites of TRAFproteins vary a lot, and all of the TRAF proteins except for TRAF1possess a RING-finger motif at the N-terminal site thereof. ARING-finger is essential for the TRAF-mediated signal transduction. Inthe previous study, when the genes encoding all six types of TRAFproteins were knockout in mouse by gene targeting, various phenotypeswere observed, suggesting that TRAFs have significantly variousbiological functions (Ha, H., Curr Protoc Immunol. Chapter 11:Unit11.9D, 2009).

A Ca²⁺-dependent transcription factor known as nuclear factor ofactivated T cells (NFATs) regulates not only T cells but also varioustypes of growth factors and cytokines. Also, it regulates cell-to-cellinteraction molecules essential for morphogenesis, development, andother functions in various types of cells and organs. Recently, NFAT isshown to be an important factor in induction of specific geneticprograms that regulate differentiation to T_(H) lineage and effector orregulatory functions of T_(H) cells, via the transcriptional regulationof T_(H) lineage-specific transcription factors such as T-bet (T_(H)1),GATA-3 (T_(H)2), RORγt (T_(H)17), and Foxp3 (T_(reg)). In addition, ithas been reported that NFAT family regulates transcription of variouscytokines and cytokine receptors thereof (Macian, F. Nat Rev Immunol. 5,472-484, 2005).

One of the most important factors determining the fate of CD4⁺ T cell iscytokine milieu during the TCR-mediated activation of naive CD4⁺ T cell.The major signaling pathway triggered by cytokines is an activation ofthe signaling transducer and activator of transcription (STAT) familyproteins. The STAT proteins play an essential role in differentiationand expansion of T_(H) cells. In particular, IFN-γ/NFAT1/STAT-1 pathwayactivates T-bet which is the master transcription factor specifying theT_(H)1 lineage, while NFAT1/STAT-3 pathway activates RORγ which is themaster transcription factor specifying the T_(H)17 lineage. Also,IL-4/NFAT2/STAT-6 pathway activates GATA-3 which is the mastertranscription factor specifying the T_(H)2 lineage, and NFAT1/STAT-5pathway activates Foxp3 which is the master transcription factorspecifying the T_(reg) lineage. These complex intracellular signalingcascades play an important role in determining the differentiation ofT_(H) cell and functions thereof.

Naive CD4 T cell can differentiate into four different types of T cellssuch as T_(H)1, T_(H)2, T_(H)17, and induced T_(reg) cells. Each of theT cells has different function and among them, T_(H)2 cell whichproduces IL-4, IL-5, IL-9, IL-10, IL-13, and IL-25 mediates immuneresponse to parasites such as helminthes. Also, T_(reg) cell is involvedin regulation of immune response and plays an important role inmaintaining self-tolerance. Increasing the number of the T_(reg) cellsor promoting a suppressive function of T_(reg) cells are known to beimportant factors in treatment of autoimmune diseases and prevention ofallograft rejection (Jinfang Zhu et al., Blood, 112, 1557-1569, 2008).

DISCLOSURE Technical Problem

Accordingly, the present inventors have identified a novel epitope ofAITR which can convert various T cells to T_(H)2 cells. Also, thepresent inventors have confirmed that an antibody that specificallybinds to the epitope can effectively convert T cells into T_(H)2 cellsand it can also promote TGF-β production in T_(reg) cells. Overall, itwas confirmed that the antibody of the present invention can be used forT_(H)2 cell conversion and for the prevention or treatment of autoimmunedisease, thereby completing the present invention.

Technical Solution

One object of the present invention is to provide a polypeptide which isan epitope, represented by an amino acid sequence of SEQ ID No.2, ofactivation-inducible tumor necrosis factor receptor (AITR) forconverting T cells to T_(H)2 cell.

Another object of the present invention is to provide an antibodyspecifically recognizing the epitope of AITR, represented by SEQ IDNo.2.

Another object of the present invention is to provide a method forpreparing the antibody.

Another object of the present invention is to provide a polynucleotideencoding the epitope or antibody, an expression vector comprising thepolynucleotide, and a transformant introduced with the vector.

Another object of the present invention is to provide a compositioncomprising the antibody for converting T cell T_(H)2 cell.

Another object of the present invention is to provide a method forconverting T cell to T_(H)2 cell, comprising the step of treating T cellwith the antibody.

Another object of the present invention is to provide a pharmaceuticalcomposition comprising the antibody for preventing or treatingautoimmune diseases.

Another object of the present invention is to provide a method fortreating autoimmune diseases using the antibody.

Advantageous Effects

The present invention has identified a new epitope of AITR, representedby SEQ ID No.2, which can convert T cell to T_(H)2 cell. Also, it wasconfirmed that an antibody that can specifically recognize the epitopecan effectively convert T cell to T_(H)2 cell. Accordingly, the newlyidentified epitope of the present invention for converting to T_(H)2cells can be used for treatment of various diseases or research purposethat needs T_(H)2 cells. Furthermore, the antibody of the presentinvention not only effectively converts T cells to T_(H)2 cells, butalso increases the suppressive function of T_(reg) cells effectively.Therefore, the antibody can be effectively used for preventing ortreating a disease like autoimmune disease which requires enhancement ofthe suppressive function of T_(reg) cells.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1D demonstrate the process of generating anti-AITR mAbs fromthe human antibody library. FIG. 1A shows the Coomassie blue-stained gelshowing the presence of anti-AITR Fab clones selected by phagedisplaying; and bivalent forms of anti-AITR mAbs that were generatedfrom transiently transfected HEK293. FIG. 1B shows flow cytometryanalysis results demonstrating the binding of anti-AITR mAbs to AITR inAITR-overexpressed Jurkat cells that were stained with PE-conjugatedanti-AITR mAbs. FIG. 1C shows the proportion of AITR-expressing cells(%) in a population of CD4⁺, CD8⁺, and CD19⁺ cells. PBMCs werestimulated with anti-CD3/IL-2 or lipopolysaccharide (LPS) for 2 days andstained with PE-conjugated anti-AITR mAb (clone A41). FIG. 1D shows theexpression of AITR in activated CD4⁺ T cells. The purified CD4⁺ T cellswere stimulated with anti-CD3/IL-2 for 3 days. AITR expression in theactivated CD4⁺ T cells was detected with PE-conjugated anti-AITR mAbs.

FIGS. 2A-2B show the expression of AITR in CD4⁺ T cells in anactivation-dependent manner. Isolated PBMCs were stimulated withanti-CD3/IL-2 or LPS for 2 days. FIG. 2A shows the stimulated cellsstained with PE-conjugated anti-AITR mAb (clone A41) in a test group ofCD4⁺, CD8⁺, and CD19⁺ cells. Expressions of AITR (arrow pointing) inCD4⁺ T cells (upper panels), CD8⁺ T cells (middle panels), and CD19⁺cells (down panels) were detected by staining both of the non-treatedgroup or activated group of cells. FIG. 2B shows the stimulated cellsstained with PE-conjugated anti-AITR mAb in a group of CD11c⁺CD14⁺,BDCA-2⁻ and CD56⁺ cells. Expressions of AITR were detected by stainingthe non-treated group (left panels) or activated group (right panels).The representative flow cytometric profiles are shown as dot plotdiagrams. The proportion of AITR-expressing cells in each cellpopulation is indicated. One of the results from three independentexperiments is shown in this figure.

FIGS. 3A-3C show the epitope mapping of the anti-AITR mAbs of thepresent invention. FIG. 3A shows a schematic diagram of full-length AITRand recombinant GST protein lysates used for antibody production andepitope mapping. Top: an intracellular domain (ICD) and an extracellulardomain (ECD) of AITR. Bottom: twelve AITR recombinants R1, R2, and R3 toR12 (R3-R12). FIG. 3B shows western blotting analysis demonstrating aspecific binding of anti-AITR mAbs to each fragment of AITR-ECD.Fragments of AITR-GST proteins are shown by Coomassie blue staining (Topand left panel). Molecular weight marker and recombinant GST proteinsize are indicated on the left of panel. FIG. 3C shows the position ofeach epitope for anti-AITR antibodies (A27, A35, A41, B32, and B62)within the amino acid sequence of AITR-extracellular domain (ECD) (SEQID No.1).

FIGS. 4A-4B demonstrate the induction of differentiation of CD4⁺ T cellsinto different phenotypes by anti-AITR mABS which recognize differentfragments of AITR-ECDs of the present invention. FIG. 4A shows theanalysis of cytokine secretion through human T_(H)1/T_(H)2 cytokine beadarray and IL-17A ELISA assay. The purified CD4⁺ T cells were stimulatedwith anti-CD3/anti-AITR mAbs (clones A27, A35 or A41) for 3 days. FIG.4B shows the proportions of cytokine-secreting cells, i.e., IL-4⁺,IFN-γ⁺, and IL-17A⁺ cells (%). Purified CD4⁺ T cells were stimulatedwith anti-CD3/IL-2 for 3 days and treated with anti-AITR mAb or AITRligand for additional 7 days. The cultured cells were stained forendogenous IL-4, IFN-γ, and IL-17A, which were then analyzed by flowcytometry. Then the proportion of IL-4+, IFN-γ⁺, and IL-17A⁺ cells (%)were calculated. The bar graphs (mean±SD) show the average value of datafrom three independent experiments, single asterisk, *p<0.05.

FIG. 5 shows the induction of cell division in CD4⁺ T cells by anti-AITRmAbs. CFSE-labeled CD4⁺ T cells were seeded in a flat-bottomed 96-welltissue culture plate coated with anti-CD3/IL-2 and treated withanti-AITR mAbs for 3 days. Histograms are shown in a resolution able toshow up to five cycles of cell divisions by flow cytometry. Theexperimental result shown is based on one of the three independentexperiments.

FIGS. 6A-6C show the induction of expression of T_(H) lineage-specificmarked in CD4⁺ T cells. Purified CD4⁺ T cells were stimulated withanti-CD3/IL-2 for 3 days (A), and treated with anti-AITR mAbs (B) orAITR ligand (C) for additional 7 days. The cultured cells were stainedfor endogenous IL-4, IFN-γ, and IL-17A, and the surfaces thereof werestained with PE-conjugated anti-AITR mAb (clone A41). Then the cellswere analyzed by flow cytometry. The experimental result shown is basedon one of the three independent experiments.

FIG. 7A-7D demonstrate that the anti-AITR monoclonal antibodies of thepresent invention recognizing different ECDs of AITR recruit differentsignaling proteins in CD4⁺ T cells, thereby demonstrating differentactivities. Purified CD4⁺ T cells were stimulated with anti-CD3/IL-2 for3 days and were further treated with anti-AITR mAbs for 2, 6, 12, and 24hr. FIG. 7A shows the western blotting analysis demonstrating theexpression of TRAFs. After 24 hr of culturing, the cultured cells werelysed and immunoprecipitated with anti-AITR mAbs (clones A27, A35 andA41) and Protein G. Then the immunoprecipitates were immunoblotted withanti-TRAF1, anti-TRAF2, anti-TRAF3, anti-TRAF5, anti-TRAF6, or anti-AITRmAb (clones A27, A35 and A41). FIG. 7B shows the western blottinganalysis demonstrating the expression of NFAT1/2, p-p38, p-ERK1/2,p-JNK1/2, and p-NF-KB. The cells were treated with anti-AITR mAbs forthe indicated times and lysed. Then the cell lysates were analyzed bywestern blotting using the antibodies against NFAT1/2, p-p38, p-ERK1/2,p-JNK1/2, and p-NF-KB. The expression level of the target proteins wasnormalized to β-actin expression level. FIG. 7C shows the flow cytometryanalysis showing the expression of STATs. After 24 hours of culturing,the cells were analyzed by flow cytometry using the antibodies againstp-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5 and p-STAT-6. FIG. 7D shows theflow cytometry analysis demonstrating the expression of STAT proteinsand master transcription factors. After 24 hr of culturing, the cellswere stained with T-bet, GATA-3, or RORγt antibody and analyzed by flowcytometry. Expression of STAT proteins and master transcription factors(filled histograms, arrow pointing) are shown. Numerical valuesindicated in each panel represent an average of the data (mean±SD) fromthree independent experiments (compared with control, open histograms).

FIG. 8 shows the recruitment of TRAF protein and activation of signaltransduction molecule by anti-AITR mAb in CD4⁺ T cell and T_(reg) cell.

FIG. 9 shows the activation of STAT protein and master transcriptionfactor by anti-AITR mAb in CD4⁺ T cell and T_(reg) cell.

FIG. 10 shows the switching effect of anti-AITR mAbs in CD4⁺ T cells.Purified CD4⁺ T cells were stimulated with anti-CD3/IL-2 for 3 days foractivation. Then the cells were further treated with anti-AITR mAbs foradditional 7 days. Subsequently, the treated cells were washed andre-stimulated with media comprising the antibodies having different orsame epitopes for 7 days. The cells were stained for endogenouscytokines IFN-γ, IL-4, and IL-17A. Then the effector phenotypes wereexamined. Also, the number of cell populations was calculated. Datashown are the average of three independent experimental results(mean±SD), single asterisk, *p<0.05 (compared to other groups).

FIG. 11 shows the switching effect of anti-AITR mAbs in CD4⁺ T cell.

FIGS. 12A-12D show the AITR-mediated conversion of human T_(reg) cell toTH cell and enhancement of immune suppressing function of T reg cell byA41. The sample of CD4⁺ T cells isolated from heal thy subjects andcancer patients were treated with anti-CD3/IL-2 for 2 days foractivation. FIG. 12A shows the analysis of the effector phenotypes ofcytokines. CD4⁺CD25^(high) T cells isolated from heal thy subjects weretreated with anti-AITR mAbs for 7 days. The cultured cells were stainedfor endogenous cytokines IFN-γ, IL-4, and IL-17A, and the effectorphenotypes were examined by flow cytometry. FIG. 12B shows themeasurements of the expression level of cytokines. The cell cultureswere collected at the indicated times and the level of TGF-, IFN-γ,IL-4, and IL-17A cytokines therein was measured. FIG. 12C shows themeasurements of the expression level and mean fluorescence intensity(MFI) of master transcription factors. The isolated CD4⁺CD25^(high) Tcells were stimulated with anti-AITR mAbs. The cell cultures werecollected at the indicated times and stained with the antibodies againstT-bet, RORγt, GATA-3 and Foxp3. The expression level and meanfluorescence intensity (MFI) of these transcription factors weremeasured by using flow cytometry. FIG. 12D shows the analysis of theeffector phenotypes of master transcription factors. The CD4⁺CD25^(high)T cells isolated from cancer patients were stimulated with anti-AITRmAbs for 7 days. Subsequently, the treated cells were stained forendogenous cytokines IFN-γ, IL-4, and IL-17A, and the effectorphenotypes thereof were analyzed by flow cytometry. The cells weresorted by using flow cytometry, which were then treated with anti-AITRmAbs for 7 days. The level of endogenous cytokines IFN-γ, IL-4, andIL-17A in the cultured cell was measured by using flow cytometry. Thenumber of cell populations was calculated. The data represents theresults from three independent experiments (mean±SD). *p<0.05 (comparedto other groups)

FIGS. 13A-B show the confirmation of AITR-mediated conversion of humanTreg cells isolated from healthy subjects to T H cells. CD4⁺ T cellspurified from healthy subjects were stimulated with anti-CD3/IL-2 for 5days. FIG. 13A shows the expression of AITR and Foxp3 in CD4⁺CD25^(high)T cells (R1) and CD4⁺CD25− T cells (R2). CD4⁺CD25^(high) T cells (R1)and CD4₊CD25− T cells (R2) were sorted by FACS sorter which is a flowcytometry. Then the expression of AITR and Foxp3 was detected in thesorted cells. FIG. 13B shows the analysis of effector phenotype ofCD4⁺CD25^(high) T cells and CD4⁺CD25− T cells. The sortedCD4⁺CD25^(high) T cells and CD4⁺CD25− T cells were stimulated withanti-AITR mAbs. The stimulated cells were stained for endogenouscytokines IL-4, IFN-γ, and IL-17A and were examined for effectorphenotype by flow cytometry. One of the three independent experimentalresults is shown.

FIGS. 14A-14B show the recruitment of TRAFs and activation of othersignal molecules in CD4⁺CD25^(high) T cells by anti-AITR mAbs. Thesorted CD4⁺CD25^(high) T cells were stimulated with anti-AITR mAbs for6, 12, or 2 4 hours. FIG. 14A shows the western blotting analysis ofCD4⁺CD25^(high) T cells for the expression of TRAF proteins. After 24hours of culturing, cell lysates were prepared, which were thenimmunoprecipitated with protein G. The immunoprecipitates wereimmunoblotted with anti-TRAF1, 2, 3, 5 and 6, or anti-AITR mAb. FIG. 14Bshows the western blotting analysis of CD4⁺CD25^(high) T cell lysatesprepared at the indicated times for the expression of NFAT1/2, p-p38,p-ERK1/2, p-JNK1/2, p-NF-KB, or -actin. Cell lysates were prepared atthe indicated times and immunoblotted with NFAT1/2, p-p38, p-ERK1/2,p-JNK1/2, p-NF-KB, or -actin. The results were normalized according tothe level of -actin expression. Data shown here are representative ofthree independent experiments.

FIGS. 15A and B show the induction and activation of STAT proteins andmaster transcription factors in CD4⁺CD25^(high) T cells by anti-AITRmAbs. The sorted CD4⁺CD25^(high) T cells were stimulated with anti-AITRmAbs. FIG. 15A shows the analysis of STAT protein expression. After 24hours of culturing, the cultured cells were analyzed by flow cytometryusing the antibodies against p-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5 andp-STAT-6. Expressions of STAT proteins are shown as filled histograms(arrow pointing) in comparison to the control group (unfilledhistogram). Data are representative of three independent experiments(mean±SD) FIG. 15B shows the flow cytometry analysis of T-bet, GATA-3,RORγt and Foxp3. Culture cells were collected at the indicated times andstained with T-bet, GATA-3, RORγt and Foxp3 antibodies, and thenanalyzed by flow cytometry. One of the three independent experimentalresults is shown.

BEST MODE

As one aspect, the present invention provides a polypeptide defined byan amino acid sequence of SEQ ID No.2, which is an epitope ofactivation-inducible tumor necrosis factor receptor (AITR), forconverting T cell to type 2 helper T (T_(H)2) cell.

As used herein, “activation-inducible tumor necrosis factor receptor(AITR)” refers to a type of receptor belonging to TNFR superfamily, andcan be interchangeably used with glucocorticoid-induced TNFR-relatedprotein (GITR), tumor necrosis factor receptor superfamily member 18(TNFRSF18), or CD357. The AITR may preferably be human AITR, but is notlimited thereto. Information on AITR can be obtained from a well-knowndatabase such as NCBI GenBank. For instance, the information on AITR maybe found by searching with NCBI Reference Sequence: NP 004186, but isnot limited thereto. The AITR is expressed constitutively at high levelin activated T cell or T_(reg) cell.

As used herein, the term “epitope” refers to a site on antigendetermining the antigen specificity, and can be interchangeable usedwith antigen determining group or antigen determining site. For thepurpose of the present invention, the epitope refers to the sitecorresponding to 20^(th) to 30^(th) amino acids of AITR-ECD, which canconvert T cell to T_(H)2 cell or enhance suppressive function of T_(reg)cell, but is not limited thereto as long as the sequence has the sameconverting activity. Also, the scope of the epitope may include thesequence having at least 80%, 90%, 95%, 98%, or 99% sequence homology tothe sequence of 20^(th) to 30^(th) amino acids of AITR-ECD withoutlimitation, as long as it has the same activity as the above-specifiedsequence. The sequence of 20^(th) to 30^(th) amino acids of AITR-ECD isshown in FIG. 3C and Table 2, and named as SEQ ID No.2. The presentinventors identified for the first time that an epitope represented byan amino acid sequence of SEQ ID No.2 is the part that can induce theconversion of T cell T_(H)2 cell or enhance the suppressive function ofT_(reg) cell. In an effort to identify the site that can specificallyconvert T cell to T_(H)2 cell in AITR-ECD constituted of 139 amino acids(SEQ ID No.1), the present inventors have confirmed that when thesequence of 20th to 30th amino acids represented by SEQ ID No.2 is usedas an epitope, it can specifically convert T cell to T_(H)2 cell. On theother hand, for the antibodies that recognize other sequences adjacentto 20^(th) to 30^(th) amino acids as an epitope, they did not show theconverting activity to T_(H)2 cell, and thus it was identified that thesequence of 20^(th) to 30^(th) amino acids of the AITR-ECD is highlyspecific for T cell conversion to T_(H)2 cell (Experimental Examples 2and 4 to 8).

As used herein, “conversion of T cell to type 2 helper T (T_(H)2) cell”refers to converting T cell to T_(H)2 cell through antibody binding to aspecific epitope of AITR represented by SEQ ID No.2 or non-antibodyprotein specifically binding to the same. Preferably the conversionrefers to the conversion of T cell to T_(H)2 producing IL-4 through anantibody binding to the epitope of AITR in T cell represented by SEQ IDNo.2, but is not limited thereto. The conversion of T cell to T_(H)2 canbe identified by observing the expression or secretion of T_(H)2cell-specific marker, for example cytokines such as IL-4; activation ofIL-4/NFAT-2/STAT-6 signal transduction pathway; and expression oractivation of transcription factor such as GATA-3. The T cell refers toa T cell expressing AITR, and for example it may be the activated Tcell. In one example of the present invention, it was identified thatCD4⁺ T cell and regulatory T cell expressed AITR when stimulated byanti-CD3/IL-2 or LPS. The T_(H)2 cell is an immune cell secreting 1L-4.T_(H)2 cell mediates immune response to parasites. Therefore, apolypeptide of the present invention which is an epitope of AITRrepresented by SEQ ID No.2 that can convert T cell can be effectivelyused in the development of antibody for converting the cells to T_(H)2cells. In the conversion of T cell to TH2 cell, the T cell includesvarious types of T cells such as CD4⁺ T cell, T_(H)1 cell or T_(H)17cell, but is not limited thereto. In one Example of the presentinvention, it was confirmed that CD4⁺ T cell can be converted to T_(H)2(FIGS. 4A-4B and 6A-6C), and also T_(H)1 cell or T_(H)17 cell withdesignated cell fate can be converted to T_(H)2 cell by A41 which is anantibody that binds to the epitope of SEQ ID No.2 of the presentinvention (FIGS. 10 and 11). Thus, epitope of AITR with SEQ ID No.2 isidentified as an important site for conversion of various T cells toT_(H)2 cells.

As used herein, “suppressive function of T_(reg) cell” refers to afunction of suppressing immune response, that is, inhibiting theactivity, proliferation, or function of various types of immune cellssuch as CD8⁺ T cells, natural killer (NK) cells, B cells, but is notlimited thereto. The T_(reg) cell refers to a type of T cell that candown regulate immune system. Also, a regulatory T cell can beinterchangeably used with T_(reg) cell, CD4⁺CD25^(high) cell, orCD4⁺CD25^(high) Foxp3⁺ cell. The T_(reg) cell comprises both of naturalor constitutive regulatory T cell and adaptive or inducible regulatory Tcell. The suppressive function of T_(reg) cell can be enacted throughvarious pathways. To be specific, the suppressive function can beenacted by 1) suppressing immune response through secreting inhibitorycytokines such as IL-10, IL-35, or TGF−, 2) binding to CD80/CD86expressed in effector T cell or APC through CTLA4 and inhibiting thefunctions thereof, or 3) preventing IL-2 from binding to a receptor ofnaive T cells by introducing an alternative IL-2 receptor that hassignificantly higher affinity to IL-2 than a natural IL-2 receptorpresent in native T cells, but is not limited thereto. The antibody ofthe present invention that specifically recognizes the epitope of SEQ IDNo.2 acts to enhance the suppressive function of T_(reg) cell. Theenhancement of suppressive function can be confirmed by using aconventional method in the art, and the example of such method includesmonitoring of the activation of signal transduction pathway such asSTAT-5 or the increase of secretion of cytokines such as TGF− whichfunctions as a main regulatory factor in the suppressive function ofT_(reg) cell.

In one example of the present invention, it was confirmed that activatedT cell was differentiated into T_(H)2 cell by A41 which is arepresentative antibody of the present invention that specificallyrecognizes the epitope of SEQ ID No.2. However, other AITR-specificantibody recognizing the site other than the epitope of SEQ ID No.2 didnot show the above converting activity. To be specific, A27 antibodyrecognizing the sequence of 56^(st) to 65^(th) amino acids and A35antibody recognizing the sequence of to amino acids could notdifferentiate T cell to T_(H)2 cell, even though these epitope siteswere close to the sequence of 20^(th) to 30^(th) amino acids of AITR-ECD(FIGS. 3A-3C, 4A-4B and 6A-6C). In addition, even after the T cell wasconverted to the cells that secrete a specific cytokine throughtreatment with A27 or A35, the cell could be re-converted toIL-4-secreting cell, when treated with A41 antibody recognizing theepitope of SEQ ID No.2 of the present invention (FIGS. 10 and 11). Theseresults suggest that the epitope of SEQ ID No.2 is significantlyimportant site in cell conversion to T_(H)2 cell. Also, in one exampleof the present invention, a molecular mechanism behind the conversion toT_(H)2 cell through an epitope of SEQ ID No.2 was investigated. NFAT2 isinvolved in the conversion to T_(H)2 cell and it increasesphosphorylation level of STAT-5 and STAT-6 proteins (FIGS. 7A-7D to 9).In addition, it was observed that a representative antibody of thepresent invention, A41, increased the level of GATA-3 which is a T_(H)2cell-specific marker (FIG. 7D), thereby confirming that T cell could beefficiently converted to T_(H)2 cell by the present antibody. Also, whenTreg cell was treated with a representative antibody of the presentinvention, A41, the secretion of inhibitory cytokine such as TGF− wassignificantly increased (FIG. 12B) and the phosphorylation of STAT-5 waspromoted (FIGS. 15A-15B). These results suggest that when the antibodyof the present invention acts specifically to AITR of T_(reg) cell, thesuppressive function of T_(reg) cell can be effectively enhanced.

As another aspect, the present invention provides an antibodyspecifically recognizing an epitope of activation-inducible tumornecrosis factor receptor (AITR), wherein the epitope is represented bySEQ ID No.2.

As used herein, the term “antibody” refers to a protein molecule thatacts as a receptor recognizing an antigen specifically, comprising animmunoglobulin molecule that responses to a specific antigenimmunologically. Also, the antibody comprises all of polyclonalantibody, monoclonal antibody, whole antibody, and antibody fragment. Inaddition, the scope of antibody includes a chimeric antibody (forexample, humanized murine antibody) and bivalent or bispecific molecule(for example, bispecific antibody), diabody, triabody, and tetrabody.The whole antibody is constituted of two full-length light chains andtwo full-length heavy chains, wherein each of the light chains isconnected with heavy chains through disulfide binding. The wholeantibody comprises IgA, IgD, IgE, IgM, and IgG. IgG comprises IgG1,IgG2, IgG3, and IgG4 as subtypes thereof. The antibody fragment refersto a fragment capable of binding to antigen, and it comprises Fab, Fab′,F(ab′)2, and Fv. The Fab is constituted of variable regions of lightchain and heavy chain, constant region of light chain, and the firstconstant region of heavy chain (CH1 domain), having one antigen-bindingsite. Fab′ is different from Fab in that it has a hinge region whichcomprises more than one cysteine residue at C-terminal of heavy chainCH1 domain. F(ab′) 2 antibody is formed through disulfide bonding ofcysteine residues in the hinge region of Fab′. A variable fragment (Fv)refers to a minimized antibody fragment having heavy chain variableregion and light chain variable region. In a double stranded Fv (dsFv),heavy chain variable region and light chain variable region areconnected through disulfide bonding while in a single stranted Fv(scFv), heavy chain variable region and light chain variable region arecovalently bonded through peptide linker. These antibody fragments canbe obtained by treating the antibody with protease (for example, when awhole antibody is restriction digested with papain, Fab is obtained andwhen restriction digested with pepsin, F(ab′)2 fragment can beobtained). Preferably, the antibody fragment can be generated by usinggenetic recombination technique.

As used herein, the term “monoclonal antibody” refers to an antibodymolecule that has been obtained from a substantially identical antibodyclone, which shows single-binding specificity and affinity for aspecific epitope.

Typically, immunoglobulin has heavy chain and light chain and each ofthe heavy chain and light chain possesses constant region and variableregion (also known as domains). A variable region of light chain andheavy chain comprises three variable regions and four framework regionscalled complementarity-determining region (hereinafter referred to as“CDR”) The CDR mainly acts to bind to an epitope of antigen. CDRs ofeach chain is conventionally called CDR1, CDR2, and CDR3 successivelystarting from the N-terminal, and also they are distinguished by thechain at which they are located.

As used herein, the term “human antibody” refers to a molecule derivedfrom human immunoglobulin, in which all of the amino acid sequencesconstituting the antibody including complementarity-determining regionsand framework regions are composed of amino acid sequences of humanimmunoglobulin. Human antibody is conventionally used for treating humandiseases, which has more than three potential advantages. First, humanantibody interacts more favorably with human immune system, and thus itcan destroy the target cell more efficiently, for example throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). Secondly, human immune system doesnot recognize the human antibody as a foreign substance. Thirdly, whensmaller dose of drug was administered at lower frequency, half-life ofhuman antibody in human circulation is similar to that ofnaturally-occurring antibodies. In one example of the present invention,it was confirmed that a human monoclonal antibody specificallyrecognizing the epitope of SEQ ID No.2 demonstrates a strong affinitytoward AITR and thus can effectively convert T cell to T_(H)2 cell andenhance the suppressive function of T_(reg) cells (FIGS. 1A-1D to15A-15B). The heavy chain and light chain domains of the presentantibody having the activity of converting T cell to cell and enhancingthe suppressive function of T_(reg) cells are all human-derived showinglower rate of immunogenicity, and thus the antibody can be effectivelyused for treating autoimmune disease.

As used herein, “antibody specifically recognizing an epitope ofactivation-inducible tumor necrosis factor receptor (AITR) representedby SEQ ID No.2” refers to an antibody that can convert T cell to T_(H)2cell by specifically recognizing an epitope of SEQ ID No.2 or enhancethe suppressive function of T_(reg) cell, but is not limited thereto.The antibody particularly enhances the suppressive function of T_(reg)cells, for example, production of inhibitory cytokine, TGF-β, and thusit can be effectively used for preventing or treating autoimmunedisease; and for enhancing immunity.

The antibody specifically recognizing the epitope of SEQ ID No.2 maypreferably comprise a heavy chain variable region comprising heavy chaincomplementarity-determining region 1 (CDR1) represented by SEQ ID No.6;heavy chain CDR2 represented by SEQ ID No.7; and heavy chain CDR3represented by SEQ ID No.8, and a light chain variable region comprisinglight chain CDR1 represented by SEQ ID No.10; light chain CDR2represented by SEQ ID No.11; and light chain CDR3 represented by SEQ IDNo.12. More preferably, the antibody may comprise an amino acid sequenceof heavy chain variable region represented by SEQ ID No.5 and an aminoacid sequence of light chain variable region represented by SEQ ID No.9,but is not limited thereto. In one example of the present invention, ahuman monoclonal antibody comprising the amino acid sequence of heavychain variable region represented by SEQ ID No.5 and the amino acidsequence of light chain variable region represented by SEQ ID No.9 wasnamed as A41. In addition, the polynucleotide encoding the antibody maycomprise a nucleotide sequence of a heavy chain variable regionrepresented by SEQ ID No.13; and a nucleotide sequence of a light chainvariable region represented by SEQ ID No.14, but is not limited thereto.

In addition, if the antibody of the present invention comprises aconstant region, the constant region may be derived from IgG, IgA, IgD,IgE, and IgM or a combination or hybrid thereof.

As used herein, the term “combination” refers to, when forming a dimeror polymer, a combination of a polypeptide coding for single-chainimmunoglobulin constant region of same origin with single-chainpolypeptide of different origin. For example, the dimer or polymer canbe formed with more than two constant regions selected from the groupconsisting of constant regions of IgG, IgA, IgD, IgE, and IgM.

As used herein, the term “hybrid” refers to the presence of more thantwo immunoglobulin heavy chain constant regions of different originswithin a single-chain immunoglobulin heavy chain constant region. Forexample, a hybrid of domains can be formed with 1 to 4 domains selectedfrom the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD,IgE, and IgM. Meanwhile, a combination or hybrid of heavy chain constantregions of the subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 isalso possible. The combination and hybrid are the same as describedabove.

Also, if the antibody of the present invention comprises a light chainconstant region, the light chain constant region may be derived fromlambda(A) or kappa(K) light chain.

In one example of the present invention, A41 that specifically binds tothe epitope of AITR with SEQ ID No.2 was selected from a human antibodylibrary (Experimental Example 1), and their specific binding to AITR wasconfirmed (FIGS. 1A-1D). In addition, the antibody recognition sites onAITR-ECD were identified (FIGS. 3A-3C and Table 2). Also, it wasconfirmed that unlike other antibodies that recognize other sites ofAITR, the above-described antibodies can convert T cell to T_(H)2 cell(FIGS. 4A-4B to 11), and furthermore, even the T cells that were alreadydifferentiated into specific T cell, the antibodies can re-convert thecell to cell (Experimental Examples 7 and 8, and FIGS. 10 and 11). Inaddition, when the antibody of the present invention acted to T_(reg)cells, it increased the production level of TGF-β in T_(reg) cells,thereby enhancing the suppressive function of T_(reg) cells and at thesame time, the phosphorylation level of STAT-5 was also increased (FIGS.12A-12D to 15A-15B). These results support that the AITR-specificantibody of the present invention that recognizes an epitope of SEQ IDNo.2 can be used for prevention or treatment of autoimmune disease thatrequires efficient conversion of T cell to T_(H)2 cell; and enhancementof suppressive function of T_(reg) cells.

As another aspect, the present invention provides a method for preparingthe antibody.

The antibody of the present invention can be easily prepared by aconventional method for production of antibody. For example, amonoclonal antibody can be prepared by generating hybridoma with Blymphocyte obtained from immunized animals (Koeher and Milstein, 1976,Nature, 256:495), and phage display technique, but is not limitedthereto. A polyclonal antibody can be easily prepared by a conventionalmethod for production of antibody, and also it can be prepared by usingan antigen comprising the epitope of the present invention.

Production of antibody library by using phage display technique is doneby obtaining an antibody gene directly from B lymphocyte withoutproducing hybridoma and expressing the antibody on the phage surface. Byusing the phage display technique, many limitations associated with theproduction of monoclonal antibody can be overcome by B-cellimmortalization. In general, a phage display technique consists of thesteps of (1) inserting oligonucleotides of random sequences to thenucleotide sequence corresponding to N-terminal of phage coat proteinpIII (or pIV); (2) expressing a fusion protein of a partial naive coatprotein and a polypeptide coded by the oligonucleotide of randomsequence; (3) treating a receptor material that can bind to thepolypeptide coded by the oligonucleotide; (4) eluting the peptide-phageparticles bound to receptors by lowering pH or using competitivemolecules for binding to the receptors; (5) amplifying a phage sampleeluted by panning within the host cells; (6) repeating the above processto obtain desired amount of phage products; and 7) determining thesequence of active peptide among the DNA sequences of phage clonesselected by panning.

Preferably, production of a monoclonal antibody of the present inventioncan be done by using phage display technique. Those skilled in the artcan easily perform each step of the preparation method of the presentinvention according to a conventional phage display technique, forexample a method described in Barbas et al. (METHODS: A Companion toMethods in Enzymology 2:119, 1991; J. Virol. 2001 July; 75(14):6692-9)and Winter et al. (Ann. Rev. Immunol. 12:433, 1994). A type of phagethat can be used for generating antibody library includes filamentousphage such as fd, M13, f1, If1, Ike, Zj/Z, Ff, Xf, Pf1, or Pf3 phage,but is not limited thereto. In addition, a type of vector that can beused for expressing heterologous gene on filamentous phage surfaceincludes a phage vector such as fUSE5, fAFF1, fd-CAT1, or fdtetDOG or aphagemid vector such as pHEN1, pComb3, pComb8, or pSEX, but is notlimited thereto. Also, a type of helper phage that can be used forproviding a wild-type coat protein which is required for the successfulreinfection of recombinant phage for amplification includes M13K07 orVSCM13 helper phage, but is not limited thereto.

The hybridoma-derived monoclonal antibody or polynucleotide encodingphage display clones of the present invention can be easily isolated andanalyzed for the sequence thereof through a conventional process. Forexample, oligonucleotide primers designed to specifically amplify theheavy chain and light chain coding region from hybridoma or phagetemplate DNA can be used. Once the polynucleotide is isolated, it can beinserted to an expression vector, which can be subsequently introducedto a suitable host cell, thereby producing a desired monoclonal antibodyfrom the transformed host cell (i.e., transformant). Therefore, themethod for preparing the antibody of the present invention may be amethod for preparing antibody comprising the step of amplifying theexpression vector comprising polynucleotide encoding the antibody, butis not limited thereto.

As another aspect, the present invention provides a polynucleotideencoding the epitope or antibody, an expression vector comprising thepolynucleotide, and a transformant introduced with the vector.

The antibody is the same as described above.

The expression vector of the present invention comprising apolynucleotide encoding the epitope or antibody is not limited to, butmay be a vector that can replicate and/or express the polynucleotide ineukaryotic cells including animal cells (for example, human, monkey,rabbit, rat, hamster, and mouse cell etc), plant cell, yeast cell,insect cell, or bacterial cell (for example, E. coli), or prokaryoticcells. Preferably, the vector may be a vector wherein the nucleotide isoperably linked to a suitable promoter for the expression thereof in ahost cell and at least one selection marker is included. For example,the expression vector of the present invention may be in a form of aphage vector, plasmid vector, cosmid vector, mini-chromosome vector,viral vector, or retroviral vector wherein the polynucleotide isintroduced.

The expression vector comprising polynucleotide encoding the antibodymay be an expression vector comprising each of polynucleotides encodingthe heavy chain or light chain of the antibody, or an expression vectorcomprising all of polynucleotides encoding heavy chain or light chain.

The transformant of the present invention wherein the expression vectoris introduced is not limited to, but may be prepared from bacterialcells such as E. coli, Streptomyces, and Salmonella typhimurium; yeastcells; fungal cells such as Pichia pastoris; insect cells such asDrosophila, Spodoptera Sf9 cell; animal cells such as Chinese hamsterovary (CHO) cells, mouse bone marrow cell SP2/0, human lymphoblastoid,COS, mouse bone marrow cell NSO, 293T, BOW melanoma cell lines, HT-1080,baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells,human retinal cell PERc.6, or plant cells by transforming the same withthe expression vector.

As used herein, the term “introduction” refers to a method oftransferring a vector comprising the polynucleotide encoding epitope orantibody to a host cell. Such introduction can be done by using variousmethods known in the art such as Calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofectamine, andprotoplast fusion. Also, transduction refers to a transfer of a targetmolecule into the cell by using viral particles through infection.Furthermore, a vector can be introduced to the host cell through genebombardment. In the present invention, the term introduction can beinterchangeably used with transfection.

As another aspect, the present invention provides a compositioncomprising the antibody for converting T cell to type 2 helper T(T_(H)2) cell.

The antibody, T cell, T_(H)2 cell, and conversion of T cell to T_(H)2cell are the same as described above. The antibody of the presentinvention specifically recognizing the epitope of AITR represented bySEQ ID No. 2 can convert T cell to T_(H)2 cell specifically, and thus acomposition comprising the antibody can be used for the conversion of Tcell to T_(H)2 cell. Especially, since the composition of the presentinvention can re-convert the already differentiated cell (e.g., T_(H)1cell or T_(H)17 cell) to T_(H)2 cell, it can be diversely used dependingon the purpose.

As another aspect, the present invention provides a method forconverting T cell to type 2 helper T (T_(H)2) cell, comprising treatingT cell with the antibody.

The antibody, T cell, T_(H)2 cell, and conversion of T cell to T_(H)2cell are the same as described above. When the T cell, for example CD4⁺T cell, T_(H)1 cell, or T_(H)17 cell, are treated with the antibody ofthe present invention, it can be effectively converted to T_(H)2 cell,and thus the antibody can be effectively used in the treatment ofdiseases that need the function of T_(H)2 cell by converting T cell toT_(H)2 cell.

As another aspect, the present invention provides a pharmaceuticalcomposition for preventing or treating autoimmune disease, comprisingthe antibody.

The antibody is the same as described above.

As used herein, the term “autoimmune disease” refers to diseases thatare directly or indirectly caused by an inappropriate immune responseagainst the subject's own antigen. Type of autoimmune diseases is notlimited but may include rheumatic arthritis, systemic scleroderma,systemic lupus erythematosus, atopic dermatitis, psoriasis, areataalopecia, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome,Guilliain-Barre syndrome, chronic thyroiditis, multiple sclerosis,polymyositis, ankylosing spondylitis, encephalomyelitis, fibrositis, orpolyarteritis nodosa.

As used herein, the term “prevention” refers to all actions thatsuppress or delay the onset of autoimmune disease by administering acomposition.

As used herein, the term “treatment” refers to all actions that canalleviate or beneficially change the symptoms of autoimmune disease byadministering a composition.

The pharmaceutical composition may additionally comprise apharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier or diluent that does not inhibit a biological activity andfeature of the administered compound without irritating an organism.Types of pharmaceutical carriers suitable for a composition of liquidformulation include saline solution, sterile water, Ringer's solution,buffered saline solution, albumin solution, dextrose solution,maltodextrin solution, glycerol, ethanol, and a combination thereof, asa sterilized and acceptable carrier to the organism. If necessary, otherconventional additives such as antioxidant, buffer solution, andbacteriostatic agent can be added. Also, the pharmaceutical compositionmay be formulated into a formulation for injection such as aqueoussolution, suspension, emulsion; pill, capsule, granule, and tablet byfurther adding a diluent, dispersant, surfactant, binder, and lubricant.

The pharmaceutical composition may be in a form of various oral orparenteral formulations. The composition is formulated with conventionaldiluents or excipients, including fillers, extenders, binders, wettingagents, disintegrants, and surfactants. Solid formulations for oraladministration include tablets, pills, powders, granules, and capsules.These solid formulations are prepared by mixing one or more compoundswith at least one of excipients, for example, starch, calcium carbonate,sucrose, lactose, and gelatin. Also, other than the simple excipients,lubricants such as magnesium stearate and talc are used. In addition,types of liquid formulation for oral administration include asuspension, solution, emulsion and syrup. To this liquid formulation,not only a commonly used diluent such as water and liquid paraffin maybe added, but also various types of excipients such as wetting agents,sweetening agents, flavors, and preservatives can be added. Types offormulations for parenteral administration include sterilized aqueoussolutions, non-aqueous solutions, suspensions, emulsions, lyophilizedformulation, and suppositories. As non-aqueous solvent and suspendingagent, propylene glycol, polyethylene glycol, vegetable oils such asolive oil, and injectable esters such as ethyl oleate may be used. Thebase composition for suppository may include witepsol, macrogol, tween61, cacao butter, laurin butter, and glycerinated gelatin.

The pharmaceutical composition of the present invention may beformulated in any one of the forms selected from the group consisting oftablets, pills, powders, granules, capsules, suspension, solutions,emulsions, syrups, sterilized aqueous solution, non-aqueous solution,lyophilized formulation and suppository.

The composition of the present invention is administered in apharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” refers toan amount sufficient to treat a disease, at a reasonable benefit/riskratio applicable to any medical treatment. The effective dosage level ofthe composition may be determined by the factors including a type ofsubject, severity of condition, an age and sex of subject, a type ofvirus infected, an activity of drug, a sensitivity towards drug,duration and route of administration, excretion rate, duration oftreatment, a type of drugs used in combination with the composition, andother factors well-known in the medical field. The composition of thepresent invention may be administered alone or in combination with othermedicines, and it may be administered sequentially or simultaneouslywith conventional medicines. Also, the present composition may beadministered in single or multiple doses. In view of all of thesefactors, it is important to administer the composition in a minimumamount yielding the maximum possible effect without causing sideeffects, which can be easily determined by those skilled in the art.

In autoimmune disease, T_(reg) cell takes a key role in the suppressionof immune responses, and thus there have been many attempts to increasethe number of T_(reg) cells and enhance the suppressive function ofT_(reg) cells for treating the autoimmune disease (Kim et al., Korean JOtorhinolaryngol-Head Neck Surg, 53:737-48, 2010). In this regard, theantibody of the present invention recognizing the epitope of AITR withSEQ ID No.2 increases the production of TGF-β which is an inhibitorycytokine of T_(reg) cell having immune suppressive function. Therefore,the antibody of the present invention can be effectively used for theprevention or treatment of autoimmune disease through enhancing thesuppressive function of T_(reg) cells.

In one Example of the present invention, it was identified that theantibody of the present invention that binds to the epitope of SEQ IDNo.2 increases the production of TGF-β which is an inhibitory cytokineof T_(reg) cells, thereby confirming that the present antibody can beused for the prevention and treatment of autoimmune disease (FIG. 12B).

As another aspect, the present invention provides a method for treatingautoimmune disease, comprising administering the antibody to a subjectin need thereof.

The antibody and autoimmune disease are the same as described above.

The method for treating autoimmune disease may be a method comprisingthe step of administering the pharmaceutical composition comprising theantibody and pharmaceutically acceptable carrier in addition to asubject having autoimmune disease or suspected of having autoimmunedisease. The pharmaceutically acceptable carrier is the same asdescribed above. The method for treating autoimmune disease maypreferably be a method comprising the step of administering thecomposition comprising the antibody to a subject having autoimmunedisease.

The type of subject includes mammals and birds such as cows, pigs,sheep, chickens, dogs, and human, and is not limited as long asautoimmune disease in the subject can be treated by administration ofthe composition of the present invention.

The composition may be administered in single or multiple doses in apharmaceutically effective amount. In this regard, the composition maybe administered in a form of solutions, powders, aerosols, capsules,enteric-coated tablets or capsules or suppositories. The routes foradministration include intraperitoneal, intravenous, intramuscular,subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary andintrarectal administration, but are not limited thereto. However, sincepeptides are digestible when orally administered, the active ingredientsof a composition for oral administration need to be coated or formulatedsuch that they can be protected against degradation in stomach. Inaddition, a pharmaceutical composition may be administered through acertain apparatus capable of transporting the active ingredients into atarget cell.

The pharmaceutical composition of the present invention may comprise anantibody that specifically binds to the epitope of SEQ ID No.2. Theantibody can increase the production of TGF-β which is an inhibitorycytokine of T_(reg) cells, thereby enhancing the suppressive function ofT_(reg) cells. Therefore, through administering the pharmaceuticalcomposition comprising the antibody into the body, the onset orprogression of autoimmune disease can be prevented, thereby preventingor treating autoimmune disease.

[Mode for Invention]

Hereinafter, the present invention is described in more detail withreference to Examples. However, these Examples are for illustrativepurposes only, and the invention is not intended to be limited by theseExamples.

EXAMPLE 1: CELL ISOLATION

Peripheral venous blood samples newly collected from healthy individualsand cancer patients were treated with heparin and peripheral-bloodmononuclear cells (PBMCs) were isolated by Ficoll-paque gradientcentrifugation (GE Healthcare, Piscataway, N.J.). The isolated PBMCswere resuspended in RPMI 1640 medium containing 1% FBS for 2 days.Subsequently, naive CD4⁺ T cell subsets were collected through positiveselection using anti-CD4⁺ antibodies (Miltenyi Biotec, Gladbach,Germany) The purity of the selected CD4⁺ T cell fractions wasapproximately 97%.

In order to obtain high purity of CD4⁺CD25^(high)/− T cells, theisolated CD4⁻ T cells were stimulated with 0.1 μg/ml anti-CD3 antibodies(HIT3a; BD PharMingen, San Diego, Calif.) for 7 days, and 100 U/ml IL-2(Interleukin-2; PeproTech, Rocky Hill, N.J.) was added every 2 days.Subsequently, the cultured CD4⁺ T cells were stained with thePE-cy5-conjugated anti-CD25 antibody and FITC-conjugated anti-CD4antibody. Finally, the CD4⁺CD25^(high) T cells or CD4⁺CD25⁻ T cells wereisolated by using FACS Aria (BD Biosciences, San Jose, Calif.).

EXAMPLE 2: ANTIBODIES AND FLOW CYTOMETRY

In the flow cytometry experiment, the present inventors usedfluorochrome-conjugated antibodies against CD4, CD8, CD11c, IFN-γ,Foxp3, CTLA-4, CD62L, CD45RO, CD45RA (BD Bioscience), CD19, CD56, CD14,CD127, IL-4, IL-17A, GATA-3, T-bet, RORγt (ebioscience, San Diego,Calif.), and BDCA-2 (Miltenyi Biotec). Also, for anti-AITR antibody,biotinylated AITR-specific antibodies (A27, A35, A41, B32, and B62clones) were used. Intracellular level of cytokines was measured after 5hour-long pre-incubation with 50 ng/ml phorbol 12-myristate 13-acetate(PMA), 10 μg/ml ionomycin, and 10 μg/ml Brefeldin A (Sigma-Aldrich, StLouis. Mo.) FACSCalibur flow cytometer (BD Bioscience) was used for allof the flow cytometry analysis and WinMDI software (Ver. 2.9, Scripps.Institute) was used for data analysis.

EXAMPLE 3: PREPARATION OF SOLUBLE AITR AND AITR-OVEREXPRESSING CELL

Specific binding of human anti-AITR mAbs was measured by usingEnzyme-linked immunosorbent assay (ELISA) or FACS. In order to generatea fusion protein of a water-soluble AITR-ECD (26^(th) to 139^(th) aminoacids) and GST protein, a GST-tagged AITR-ECD expression vector(pGEX-6p-1/AITR-ECD) was constructed. First, AITR-coding sequence wasamplified by PCR using a sense primer and antisense primer shown inTable 1 and using cDNA extracted from the activated CD4⁺ T cells as aPCR template. Then the amplified AITR-coding sequence was restrictiondigested with BamHI/Xhoi, and cloned into a vector pGEX-6p-1 to generatea GST-tagged AITR-ECD expression vector (pGEX-6p-1/AITR-ECD).

TABLE 1 SEQ ID Sequence(5′→3′) No. Forward AAGCTTGGTCAGCGCCCCACCGGG 49Primer Reverse CCGGCAGAGCCGCCTTAACTCGAG 50 Primer

The pGEX-6p-1/AITR-ECD was transformed into E. coli (BL21-DE3-pLyss) andthe protein expression was induced by adding 0.20 mM IPTG. Therecombinant AITR-GST proteins expressed in E. coli were purified byrunning them through a glutathione agarose 4B bead column.

For generating AITR-overexpressing cells, the gene fragment encodingAITR-transmembrane domain (TM)-ECD was inserted in-frame with CD5Lsequence in a mammalian vector pcDNA3.1(+), i.e.pcDNA3.1(+)/CD5L-AITR-TM-ECD. Then pcDNA3.1(+)/Empty vector orpcDNA3.1(+)/CD5L-AITR-TM-ECD vector was transient transfected intoJurkat cells by electroporation. AITR expression on cell surface wasdetected by staining with biotin-conjugated anti-AITR mAb.

EXAMPLE 4: PREPARATION OF ANTI-AITR MONOCLONAL ANTIBODIES (ANTI-AITRMABS)

The pCANTAB5E phagemid vector comprising human Fab antibody cDNA librarywas transformed into TG1 E. coli cells. Subsequently, the transformantswere cultured in 2× YT broth containing 100 μg/ml ampicillin and 50μp/ml kanamycin, and then superinfected with M13K07 helper phage. Afterculturing the cells at 30° C. for 24 hours, the cell culture wascentrifuged down to collect culture supernatant only. Then thesupernatant was added with 3.3% (w/w) polyethylene glycol (Sigma) and2.3% (w/w) NaCl to precipitate recombinant phage particles obtained. Theprecipitated phage particles were resuspended in 2× YT broth.

Biopanning was performed using the above-prepared suspension as alibrary in order to select the phage specific to a human AITR-GST fusionprotein by phage display (1 to 4 times biopanning). Among therecombinant phage particles from the culture supernatant of each clone,a positive clone of human AITR-GST fusion protein was selected throughELISA.

In addition, to generate a bivalent form of antibody, Fab genes (VH andVL) obtained from phage displaying were prepared by PCR, and then clonedinto a restriction enzyme site of the expression vector pDCMV-DHFR. As aresult, an antibody gene and construct thereof of five different clones(A27, A35, A41, B32, and B62) were obtained. To generate a bivalent formof anti-human AITR mAbs, the construct of antibody gene was transientlytransfected into HEK293 and CHO cells, and the protein products werepurified from the culture supernatant by using a protein G column (GEHealthcare).

EXAMPLE 5: EPITOPE MAPPING OF ANTI-AITR MONOCLONAL ANTIBODIES (ANTI-AITRMABS)

For identifying a precise epitope recognized by the anti-AITR mAbs ofthe present invention, twelve different fragments (R1 to R12)corresponding to AITR-ECD were prepared and cloned into a GST-vector,pGEX-6p-1 (FIG. 3A)

The pGEX-6p-1/R1-R12 was transformed into E. coli cell line,BL21-DE3-pLyss, and the protein expression was induced by adding 0.20 mMIPTG. The recombinant R1-GST to R12-GST fusion proteins expressed in E.coli were purified by running them through a glutathione agarose 4B beadcolumn (Peptron). At this time, horseradish peroxidase-coupledanti-human IgG was diluted 10,000 times (Sigma Aldrich, St Louis, Mo.,USA). Also, chemiluminescence was monitored by using SuperSignal WestPico Chemiluminescence Substrate (Pierce, Rockford, Ill.).

EXAMPLE 6: CELL DIVISION AND CYTOKINE ANALYSIS

In the present invention, CD4⁺ T cells were labeled with CFSE (MolecularProbes, Invitrogen) The proliferation of CFSE-labeled CD4⁺ T cells (5×10cells/well) was stimulated by treating the cells with anti-CD3 antibody(0.1 pg/ml)/IL-2 (100 U/ml) and 5 μg/ml anti-AITR mAb for 72 hr. After72 hours of culturing, the cell-free supernatant was separated andanalyzed for cytokine level by using T_(H)2/T_(h)2 cytokines bead arraykit (BD Bioscience), human TGF-β1 Quantikine ELISA kit (R&D system,Minneapolis, Minn.), and human IL-17A ELISA kit (Abcam, Cambridge, UK).

EXAMPLE 7: ANALYSIS OF DIFFERENTIATED EFFECTOR IN CD4⁺ T CELL

Purified CD4⁺ T cells (5×105 cells/well) were stimulated with 0.1 pg/mlanti-CD3 antibody and 100 U/ml IL-2 for 3 days. Then, the cultured cellswere treated with 5 μg/ml anti-AITR mAbs or 0.1 μg/ml AITR ligand(AITRL) for another 7 days.

Subsequently, the differentiated effector T cell subsets were washedwith 1× PBS, and then cultured with a fresh medium containing 5 μg/mlanti-AITR antibody recognizing different epitope for another 7 dayswhile adding 100 U/ml IL-2 for every 2 days. Then the cultured cellswere harvested and endogenous IFN-γ, IL-4 and IL-17A were stained forthree-color flow cytometry analysis.

EXAMPLE 8: ANALYSIS OF INTREACTION BETWEEN AITR AND TRAFS

Purified CD4⁺ T cells (5×10⁵ cells/well) were stimulated with 0.1 μg/mlanti-CD3 antibody and 100 U/ml IL-2 for 2 days. The cultured cells weretreated with 5 pg/ml anti-AITR mAbs for 24 hr. Then the cells were lysedwith 100 μl RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1%Nonidet P-40, 0.25% Na-deoxycholate, 1 mM PMSF, protease inhibitors, andphosphatase inhibitors).

For immunoprecipitation, cell lysates were incubated with 20 μl of 1:1slurry of protein G-sepharose for 1 hour. Then, the precipitate waswashed with 1×PBS, and protein complexes were eluted. Western blotanalysis of eluate was performed by using anti-TRAFs (TRAF1, TRAF2,TRAF3, TRAF5, and TRAF6) (Santa Cruz Biotechnology, Santa Cruz, Calif.)or anti-AITR mAbs and using horseradish peroxidase-conjugated goatanti-rabbit IgG or anti-human IgG as a secondary antibody.

In a separate experiment, the sorted CD4⁺CD25^(high) T cells (5×10⁵cells/well) were stimulated with 5 μg/ml anti-AITR mAbs for 24 hr andthe interaction between AITR and TRAF was analyzed by using the samemethod described above.

EXAMPLE 9: ANALYSIS OF PHOSPHORYLATION OF ENDOGENOUS SIGNALING PATHWAYPROTEIN

Purified CD4⁺ T cells (1×10⁶ cells/well) were stimulated with anti-CD3antibody and IL-2 for 3 days. The cultured cells were treated withanti-AITR mAbs for 2, 6, 12 and 24 hours. Then the cells were harvestedand the suspension thereof was prepared. Levels of phospho-STATs (STAT1,STAT2, STAT3, STAT4, STAT5, and STAT6) and master transcription factors(T-bet, GATA-3, RORγt and Foxp3) were analyzed by flow cytometry. Thecells in the culture were lysed with RIPA buffer. Subsequently, totalprotein extract was resolved on 8% to 12% SDS-polyacrylamide gelelectrophoresis and immunoblotted with antibodies against NFAT1/2,p-p38, p-ERK1/2, p-JNK1/2, and p-NF-KB (Cell Signaling, Danvers, Mass.,USA). The same blot was re-probed with an anti-β-actin antibody as acontrol for protein loading.

In a separate experiment, the sorted CD4⁺CD25^(high) T cells (5×10⁵cells/well) were stimulated with 5 μg/ml anti-AITR mAbs andphosphorylation of the proteins involved in intracellular signalingpathway was analyzed by the same method described above.

EXAMPLE 10: PHENOTYPE CONVERSION OF HUMAN CD4⁺CD25^(HIGH)FOXP3⁺(T_(REG))CELL TO DIFFERENT SUBSET

Human T_(reg) cells isolated from healthy subjects and cancer patientswere sorted by the method described in Example 1. The cells werestimulated with 5 μg/ml anti-AITR mAbs for 7 days while adding 100 U/mlIL-2 every 2 days. Then the cultured cells were harvested and theexpression of AITR, CTLA-4, CD62L, CD127, CD45RO and CD4 5RA on cellsurface was confirmed. Also, endogenous IFN-γ, IL-4 and IL-17A andmaster transcription factors were stained and the cells were analyzed byflow cytometry. In addition, culture supernatants were collected and theexpression of IFN-γ, IL-4, IL-17A, and TGF-β was confirmed.

In a separate experiment, the sorted CD4⁺CD25⁻ (non-T_(reg)) cells(5×10⁶ cells/well) were stimulated with 5 μg/ml anti-AITR mAbs. Culturecells were stained for endogenous IFN-γ, IL-4 and IL-17A and thenanalyzed by three-color flow cytometry.

EXAMPLE 11: STATISTICAL ANALYSIS

All experimental data were analyzed with a statistical program calledPrism 5.0 GraphPad (San Diego, Calif.). Student's t-test was performedto determine the statistical significance of the difference between testgroups.

EXPERIMENTAL EXAMPLE 1: GENERATION OF ANTI-AITR MONOCLONAL ANTIBODIES

As described in Example 4, five Fab antibodies against AITR wereselected from a human Fab antibody library and named as A27, A35, A41,B32, and B62. The antibody A41 comprises a heavy chain variable regionof SEQ ID No.5 and a light chain variable region of SEQ ID No.9, and theantibody A27 comprises a heavy chain variable region of SEQ ID No.1S anda light chain variable region of SEQ ID No.19. The antibody B32comprises a heavy chain variable region of SEQ ID No.25 and a lightchain variable region of SEQ ID No.29, and the antibody A35 comprises aheavy chain variable region of SEQ ID No.35 and a light chain variableregion of SEQ ID No.39. The antibody B62 comprises a heavy chainvariable region of SEQ ID No.45 and a light chain variable region of SEQID No.46.

In addition, anti-AITR genes were generated by grafting V_(H) and V_(L)DNA sequences into a human IgG₁ backbone. Anti-AITR mAbs A27, A35, A41,B32, and B62, were produced by transient transfection of the antibodyconstructs into HEK293 cells (FIG. 1A). These constructs into HEK293cells (FIG. 1A). These antibodies showed similar affinity to AITR in theAITR-overexpressed Jurkat cells (FIG. 1B).

EXPERIMENTAL EXAMPLE 2: EPITOPE MAPPING OF ANTI-AITR MONOCLONALANTIBODIES

In this experiment, epitopes recognized by five human anti-AITR mAbsselected in Experimental Example 1 were identified. To confirm theepitope, AITR-GST fusion proteins named as R1 to R12 were used (FIGS.3A-3C). Immunoblotting results of RO-GST to R12-GST proteins withanti-AITR mAbs demonstrated that the five antibodies were classifiedinto 3 groups based on their recognition site of AITR, i.e., epitope.A27 and B32 specifically bound to R1 and R2 sites; A35 and B62specifically bound to R1 to R4 and R8 to R11; and A41 specifically boundto R1 to R6, R8, and R9 (FIG. 3B). As a result of investigating theepitope sites more precisely, it was confirmed that A27 and B32specifically recognized the sequence of 56^(th) to 65^(th) amino acids(AA 56-65) of AITR-ECD;

A35 and B62 specifically recognized the sequence of 41^(st) to 50^(th)amino acids (AA 41-50) of AITR-ECD; and A41 specifically recognized thesequence of 20^(th) to 30^(th) amino acids (AA 20-30) of AITR-ECD (FIG.3C and Table 2). The epitope site recognized by A41 was named SEQ IDNo.2; the epitope site recognized by A27 and B32 was named as SEQ IDNo.3; and the epitope site recognized by A35 and B62 was named as SEQ IDNo.4.

TABLE 2 Antibody Epitope SEQ ID No. A41 GTDARCCRVHT (AA 20-30) 2A27 & B32 HCGDPCCTTC (AA 56-65) 3 A35 & B62 ECCSEWDCMC (AA 41-50) 4

EXPERIMENTAL EXAMPLE 3: EXPRESSION OF AITR IN IMMUNE CELL

The expression of AITR was up-regulated after stimulation of PBMCs, butwhen PBMCs were not stimulated, mRNA of AITR was not detected. Also, theexpression of AITR was significantly up-regulated after CD3 stimulationin T cells. Furthermore, the expression of AITR was analyzed in T cells,B cells, NK cells, and monocytes by flow cytometry. The resultsdemonstrated that the human anti-AITR mAbs of the present inventionmainly recognized AITR in the activated CD4⁺ T cells which had nearly40-fold higher expression of AITR. When the antibodies were used in CD8⁺T cells, B cells, NK cells, and monocytes, neither of resting noractivated AITR was detected (FIG. 1C).

These results demonstrate that the anti-AITR mAbs of the presentinvention recognize rapidly up-regulated AITR expression in theactivated CD4⁺ T cell population, through TCR-mediated activated signal.

EXPERIMENTAL EXAMPLE 4: CO-STIMULATION OF CELL DIVISION AND CYTOKINEPRODUCTION IN DIFFERENTIATED CD4+ T CELL POPULATION BY AITR SIGNALING

After identifying that AITR is expressed mainly in the activated CD4⁺ Tcells (FIG. 1C), the role of AITR was investigated as a co-stimulatorymolecule in proliferation and cytokine secretion.

Purified CFSE-labeled CD4⁺ T cells were stimulated with immobilizedanti-CD3 and cultured in the presence of anti-AITR mAbs. Unlike whencultured with anti-CD3 only, when the cells were co-cultured withanti-AITR mAbs and anti-CD3, the proliferation of activated CD4⁺ T cellswas significantly increased (FIG. 5).

Subsequently, the culture supernatant was analyzed to identify a type ofcytokine secreted depending on the type of anti-AITR mAbs. The resultsdemonstrated that the antibodies of the present invention recognizingdifferent sites of AITR changed the level of different cytokines. To bespecific, cell treatment with a comparative antibody A27 significantlyincreased the level of T_(H)1 cytokines IL-2 and IFN-γ, whereastreatment with a comparative antibody A35, unlike with A27, increasedthe level of T_(H)17 cytokine IL-17A. On the other hand, cell treatmentwith A41 of the present invention significantly increased the level ofT_(H)2 cytokines IL-2, IL-4, and IL-5 (FIG. 4A).

In addition, the markers of phenotypic CD4⁺ T cells for the expressionof IFN-γ, IL-17A, and IL-4 in the activated CD4⁺ T cells were analyzedwhen the CD4⁺ T cells were treated with the anti-AITR mAbs or AITRL ofthe present invention. The results show that when treated with A41 ofthe present invention, the number of IL-4-producing CD4⁺ T cells wasincreased (91.5±6.21%); when treated with a comparative antibody A27,the number of IFN-γ-producing CD4⁺ T cells was increased (90.2±0.50%);and when treated with a comparative antibody A35, the number ofIL-17A-producing CD4⁻ T cells was increased (37.8±0.11%). Furthermore,the ligand thereof, i.e. AITRL promoted the production of IFN-γ inT_(H)1 CD4⁺ T cells (FIG. 4B).

Taken together, these results demonstrate that anti-AITR mAbs of thepresent invention can act as a co-stimulatory signal in CD4⁺ T cells andpromote the cell differentiation to a specific T_(H) cell and cytokinesecretion. In particular, these results support that the antibody of thepresent invention can induce different effects depending on therecognition sites of AITR. To be specific, as A41 antibody of thepresent invention recognizes the epitope of SEQ ID No.2 among manydifferent sites of AITR in cell, it can differentiate the cell toproduce a T_(H)2 cell cytokine, IL-4, among different cytokines.

EXPERIMENTAL EXAMPLE 5: INTERACTION BETWEEN AITR-FAMILY PROTEINS ANDTRAF-FAMILY PROTEINS AND DOWNSTREAM SIGNAL TRANSDUCTION

Previous studies have reported that tumor necrosis factorreceptor-associated factor (TRAF) proteins are involved in activation ofNF-KB and that downstream signaling molecules such as ERK1/2, JNK1/2,and p38 can be activated by TNFR family members. Also, cytoplasmicdomain of AITR contains acidic residues that are conserved in mouse orhuman and are involved in association of AITR with TRAF protein. AITR isknown to interact with TRAF1, TRAF2, and TRAF3, but not with TRAFS andTRAF6 (Kwon B et al., J Biol Chern 274, 6056-6061, 1999; Ha, H et al.,Curr Protoc Immunol. Chapter11:Unit11.9D, 2009). Hence, the presentinventors investigated the type of TRAF protein that AITR interacts withwhen AITR is activated by the anti-AITR mAbs of the present invention inthe activated CD4⁺ T cells.

AITR-TRAF complexes were immunoprecipitated with Protein G, from thelysate of activated CD4⁺ T cell by using anti-AITR mAbs and theimmunoprecipitates were immunoblotted with anti-TRAF antibody andanti-AITR mAbs. The results demonstrate that the antibodies of thepresent invention recognizing different sites of AITR recruit differenttypes of TRAF protein to AITR (FIG. 7A).

That is, A41 which recognizes the epitope of SEQ ID No.2 of the presentinvention recruited TRAF3 and TRAFS, whereas a comparative antibody A27which recognizes the epitope of SEQ ID No.3 of the present inventionrecruited TRAF1 and TRAF2 to AITR, and a comparative antibody A35 whichrecognizes the epitope of SEQ ID No. 4 recruited TRAF6 to AITR. Theseresults suggest that depending on the site of AITR recognized by eachantibody, different cytoplasmic signaling can be induced. Furthermore,A41 of the present invention that recruits TRAF3 and TRAFS activatedp-ERK1/2, whereas a comparative antibody A27 that recruits TRAF1 andTRAF2 activated p-JNK1/2 and p-NF-KB. On the other hand, a comparativeantibody A35 of the present invention which recruits TRAF6 activatedp-p38 and p-NF-KB (FIGS. 7A, 7B, and 8).

T cells express NFAT1, NFAT2, and NFAT3, and NFAT proteins are essentialfor producing effector cytokine when TCR is activated in T_(H) cells. Tcells also play important roles in regulating T_(H) celldifferentiation. For this reason, the present inventors investigatedwhether NFAT1 or NFAT2 can be activated when CD4⁺ T cells are treatedwith anti-AITR mAbs of the present invention. As a result, it was foundthat a comparative antibody A27 recognizing the epitope of SEQ ID No.3and A35 recognizing the epitope of SEQ ID No.4 increased the number ofactivated NFAT1 in a time-dependent manner. Unlike A27 and A35, A41 ofthe present invention increased the expression of NFAT2, but not NFAT1(FIG. 7B).

These results suggest that AITR contains a binding domain for TRAFs oradaptor cytoplasmic molecules that can activate downstream signaling.Also, the results demonstrate that depending on the site of AITRrecognized by anti-AITR mAbs, different types of TRAF proteins can berecruited, which then activates different cytoplasmic signalingpathways. Moreover, the AITR antibody specific to the epitope of SEQ IDNo.2 of the present invention not only activate specific TRAF proteins,but also show specific NFAT activities. This result suggests thatdepending on the site of AITR recognized by AITR antibody, differentNFAT translocates into the nucleus, which then induces differentNFAT-dependent gene expression.

These results support that particularly A41 of the present inventionrecognize the epitope of SEQ ID No.2 among many sites of AITR andrecruit TRAF3 and TRAFS among many different types of TRAFs, which theninduce p-ERK^(1/2)-mediated cytoplasmic signaling pathways, therebyconverting the cell to the IL-4-producing cell, wherein IL-4 is acytokine of T_(H)2 cell.

EXPERIMENTAL EXAMPLE 6: TRANSCRIPTION FACTORS AND STATS PROTEINACTIVATED DIFFERENTIATION OF CD4⁺ T CELLS WITH ANTI-AITR MABS

Previous studies have reported that signaling transducer and activationof transcription (STAT) proteins and master transcription factors areessential in fate determination of T_(H) cell and cytokine production(Hermann-Kleiter, N. & Baier G, Blood. 15, 2989-2997, 2010). Hence, thepresent inventors investigated the activities of STAT proteins andmaster transcription factors in the CD4⁺ T cells activated by anti-AITRmAbs of the present invention.

A flow cytometric analysis was performed using the antibodies againstp-STATs (p-STAT-1, p-STAT-3, p-STAT-4, p-STAT-5, and p-STAT-6) andT-bet, GATA-3, and RORyt. As a result, compared to the group treatedwith anti-CD4 and IL-2 only without anti-AITR antibody, the grouptreated with A27 showed increased number of activated p-STAT-1(52.8±0.25%), p-STAT-4 (54.2±0.19%), and T-bet (80.2±0.08%). The grouptreated with A35 showed increased number of activated p-STAT-3(63.1±1.24%) and RORγt (53.4±2.18%), and the group treated with A41showed increased number of activated p-STAT-5 (85.1±0.38%), p-STAT-6(79.2±0.12%), and GATA-3 (76.5±0.11%) (FIGS. 7C, 7D, and 9).

These results suggest that the anti-AITR mAbs of the present inventionrecognizing different sites of AITR can induce different signalingcascades mediated by AITR in CD4⁺ T cells. Especially, these resultssupport that the A41 antibody of the present invention recognizing theepitope of SEQ ID No.2 mediates the cell fate determination of T_(H)cell via a signaling pathway mediated by STAT-5, STAT-6, and GATA-3.

EXPERIMENTAL EXAMPLE 7: CONVERSION OF T CELL TO T_(H)2 CELL BY ANTI-AITRMABS

Experimental Examples 5 and 6 showed that the anti-AITR mAbs of thepresent invention specifically binding to the epitope of SEQ ID No.2regulate a specific signal transduction factor involved in T_(H) cellfate determination in CD4⁺ T cells. That is, according to the epitopesites within AITR, T cell can be converted to a totally different typeof T_(H) cell. Hence, the present inventors hypothesized that bytargeting different epitope sites recognized by anti-AITR mAbs of thepresent invention, functional phenotype of CD4⁻ T cells can beconverted. To confirm this, CD4⁺ T cells were treated with therepresentative antibodies A27, A35, and A41 which recognize threedifferent sites of AITR to determine the converting activity thereof.

The results showed that a comparative antibody A35 convertedIFN-γ-producing cells (99.2±0.21%) that were initially induced by A27, acomparative antibody specific to the epitope of SEQ ID No.2, toIL-17A-producing cells (18.8±0.23%). A41 of the present inventionconverted IFN-y-producing cells (99.2±0.21%) to IL-4-producing cells(38.7±0.32%). In addition, A35-induced IL-17A-producing cells(35.2±0.60%) were converted to IFN-γ-producing cells (82.6±1.21%) by acomparative antibody A27, and converted to IL-4-producing cells(70.5±0.62%) by A41 of the present invention. A41-induced IL-4-producingcells (98.2±0.29%) were converted to IFN-γ-producing cells (90.1±0.42%)by A27 antibody, and converted to IL-17A-producing cells (16.9±0.58%) bya comparative antibody A35 (FIGS. 10 and 11).

These results demonstrate that the antibody of the present inventionthat is specific to the AITR epitope of SEQ ID No.2 can convert the Tcell to a specific cytokine-producing cell by recognizing the specificsite of AITR, i.e. epitope of SEQ ID No.2 (AA 20-30). Furthermore, theseresults support that A41 of the present invention recognizing theepitope of SEQ ID No.2 can convert the T cell specifically toIL-4-producing T_(H)2 cell, unlike a comparative antibody A27 thatinduces cell conversion to IFN-γ-producing T_(H)1 cell, and acomparative antibody A35 that induces cell conversion toIL-17A-producing T_(H)17 cell.

EXPERIMENTAL EXAMPLE 8: ENHANCEMENT OF SUPPRESSIVE FUNCTION OFCD4⁺CD25^(HIGH) FOXP3+ (T_(REG)) CELL BY ANTI-AITR ANTIBODY

It is known that GITR or AITR acts as a co-stimulatory molecule inT_(reg) cell, and the expression thereof remains high intrinsically inT_(reg) cell even without activation. Hence, the present inventorsinvestigated the role of AITR in T_(reg) cells when stimulated byanti-AITR mAbs.

First, after stimulation with anti-CD3 and IL-2, T_(reg) cells(CD4⁺CD25^(high) cells) and non-T_(reg) cells (CD4⁺CD25⁻ cells) wereisolated from healthy subjects and cancer patients by FACS-sorter. Bothof T_(reg) cells isolated from healthy subjects and cancer patientsshowed constitutive expression of Foxp3 and AITR. Non-T_(reg) cellsshowed a lower expression of AITR than in T_(reg) cells. The number ofT_(reg) cells isolated from cancer patients was three times higher thanthat from healthy subjects.

Based on the results from the experimental example, it was determinedwhether the anti-AITR mAbs of the present invention can convert T_(reg)cells. The results showed that A41 recognizing the epitope of SEQ IDNo.2 of the present invention could not convert the T_(reg) cellsisolated from both healthy subjects and cancer patients. On the otherhand, a comparative antibody A35 converted T_(reg) cells from healthysubjects to IL-17A-producing cells (10.4±0.32%), but not the T_(reg)cells from cancer patients. Also, another comparative antibody A27converted the T_(reg) cells from both heal thy subjects and cancerpatients to IFN-γ-producing cells (93.9±0.12% and 88.2±0.43%respectively) (FIGS. 12A, 12D, and 13B). In addition, when the secretedcytokines were analyzed, A27 and A35 decreased TGF-β secretion butincreased the secretion of IFN-γ and IL-17A. On the other hand, A41 ofthe present invention increased TGF-β secretion in a time-dependentmanner which is involved in the suppressive function of Treg cells (FIG.12B). These results support that A41 of the present invention whichspecifically recognizes the epi tope of AITR of SEQ ID No.2 increasesthe secretion of TGF− in T_(reg) cells, thereby enhancing thesuppressive function of T_(reg) cells.

Furthermore, the mechanisms behind signal transduction were analyzed toinvestigate the reasons for inducing different effects in T_(reg) cells.As a result, it was observed that as shown in Experimental Example 5, acomparative antibody A27 recruited TRAF1 and TRAF2 and increased theexpression of NFAT1, thereby activating p-JNK1/2 and p-NF-kB signalingpathways. A35 recruited TRAF6 and activated p-p38 and p-NF-kB signalingpathways (FIGS. 7A-7D and 14A-14B). These results demonstrate thatcomparative antibodies A27 and A35 reduce the expression of Foxp3 butincrease the expression of T-bet or RORyt through the above signalingpathways. Through these mechanisms, a comparative antibody A27 canconvert T_(reg) cell to T_(H)1 cell, and A35 can convert T_(reg) cell toT_(H)17 cell (FIGS. 9, 12C, and 15A-15B).

On the other hand, when T_(reg) cells were treated with A41 of thepresent invention, it did not change the expression level of a T_(reg)cell marker, Foxp3, in T_(reg) cell as well as the level of GATA-3(FIGS. 9, 12C, and 15A-15B). Also, when A41 recruited TRAF protein toAITR in T_(reg) cells, it recruited TRAF3 more strongly than TRAF5, andactivated ERK1/2, NF-kB, and STAT5, thereby inducing a signaling cascadethrough NFAT1 transcription factor. Therefore, it was determined thatA41 of the present invention induces different signaling cascade fromthe antibodies recognizing other epitopes (FIGS. 9 and 14A-14B).

These results support that the antibody of the present inventionrecognizing the epitope of SEQ ID No.2 can enhance the suppressivefunction of T_(reg) cell, through different signaling pathway from thoseof other epitopes.

Based on the above description, it should be understood by those skilledin the art that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention withoutdeparting from the technical idea or essential features of the inventionas defined in the following claims. In this regard, the above-describedexamples are for illustrative purposes only, and the invention is notintended to be limited by these examples. The scope of the presentinvention should be understood to include all of the modifications ormodified form derived from the meaning and scope of the following claimsor its equivalent concepts.

The invention claimed is:
 1. A method of converting T cells to type 2 helper T (T_(H)2) cells and enhancing the function of suppressive T_(regs) in a subject having an autoimmune disease who would benefit therefrom, comprising administering to the subject a therapeutically effective amount of an antibody that specifically binds activation inducible tumor necrosis factor (AITR) wherein the antibody comprises the CDRs of SEQ ID Nos. 6, 7, 8 and 10, 11, 12, or wherein the antibody comprises the heavy chain variable domain of SEQ ID No. 5 and/or the light chain variable domain of SEQ ID No.
 9. 2. The method of claim 1, wherein the antibody comprises a heavy chain variable region comprising heavy chain CDR1 represented by SEQ ID No. 6, heavy chain CDR2 represented by SEQ ID No. 7, and heavy chain CDR3 represented by SEQ ID No. 8 and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 10, light chain CDR2 represented by SEQ ID No. 11, and light chain CDR3 represented by SEQ ID No.
 12. 3. The method of claim 1, wherein the antibody comprises a heavy chain variable region represented by SEQ ID No.
 5. 4. The method of claim 1, wherein the antibody comprises a light chain variable region represented by SEQ ID No.
 9. 5. The method of claim 2, wherein the antibody comprises a heavy chain variable region represented by SEQ ID No. 5 and a light chain variable region represented by SEQ ID No.
 9. 6. A method of treating a subject having an autoimmune disease treatable through enhancement of the suppressive function of T_(reg) cells, comprising administering to the subject a therapeutically effective amount of an antibody that specifically recognizes the polypeptide of claim 1, wherein the antibody comprises a heavy chain variable region comprising heavy chain CDR1 represented by SEQ ID No. 6, heavy chain CDR2 represented by SEQ ID No. 7, and heavy chain CDR3 represented by SEQ ID No. 8 and a light chain variable region comprising light chain CDR1 represented by SEQ ID No. 10, light chain CDR2 represented by SEQ ID No. 11, and light chain CDR3 represented by SEQ ID No.
 12. 7. The method of claim 6 wherein the antibody comprises a heavy chain variable region represented by SEQ ID No. 5 and a light chain variable region represented by SEQ ID No.
 9. 